OBJECTIVETo investigate the effect of Metformin on the proliferation and collagen synthesis of the human keloids fibroblasts as well as the effect on phosphorylation of Akt/FoxO1 signal transduction pathway.

METHODSFibroblasts of keloid were divided into control group treated with medium solution and experimental groups treated with different concentrations of Metformin. 48 h later CCK-8 assay was adopted to evaluate cell survival; Western blot was performed to detect the Akt and FoxO1 phosphorylation; and Hydroxyproline reagent kit was used to detect the collagen synthesis.

RESULTSWith different concentrations (30, 60, 90, 120 mmol/L) of Metformin, the absorbance of cultured keloid fibroblasts detected by CCK8 assay decreased by (13.30 ± 2.04)%, (22.64 ± 4.70)%, (54.00 ± 5.34)% and (63.12 ± 3.48)%. The growth of fibroblasts was suppressed by Metformin in a dose-dependent manner. It showed that the level of phoshpo-akt and phoshpo-foxOl in keloids fibroblasts in experimental groups was lower than that in the control group and the collagen synthesis were also decreased in experimental groups, all in a dose-dependent manner (P < 0.05, P < 0.01).

CONCLUSIONSMetformin can effectively inhibit the proliferation and collagen synthesis of the human keloids fibroblasts in vitro, which may be associated with the suppression of phosphorylation of Akt/FoxO1 signaling pathway

Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Keloid ; pathology ; Metformin ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects

Objective To evaluate the effect of metformin on the proliferation of keratinocytes,and to investigate its possible mechanism.Methods HaCaT human keratinocytes were divided into several groups to remain untreated (control group) or be treated with different concentrations (25,50,75,100 mmol/L) of mefformin for 24 hours (intervention groups).Subsequently,CCK8 assay was conducted to evaluate the proliferation of HaCaT cells,real-time quantitative PCR to measure the mRNA expressions of miR-21-5p and its downstream target gene PDCD4,and Western blot to detect the expression of PDCD4 protein in HaCaT cells.Statistical analysis was done by using one-way analysis of variance for multiple group comparisons and SNK-q test for paired comparisons.Results After 24-hour treatment,the proliferation of HaCaT cells was inhibited by (5.43 ± 3.67)％,(19.61 ± 6.95)％,(45.93 ± 9.56)％ and (61.91 ± 6.93)％ by metformin of 25,50,75 and 100 mmol/L,respectively,with significant differences observed in cell proliferation inhibition rates among these intervention groups (F =246.90,P ＜ 0.05).Cellular proliferative activity was similar between the control cells (0.00 ± 3.00％) and those treated with 25 mmol/L metformin,but significantly higher in the control cells than in the other 3 metformin-treated groups (all P ＜ 0.05),and significantly different between the 4 metformin-treated groups (all P ＜ 0.05).The relative mRNA expression level (2-△△Q) of miR-21-5p was 0.90 ± 0.11,0.33 ± 0.05,0.21 ± 0.07 and 0.14 ± 0.04 (F =36.99,P ＜ 0.01),while that of PDCD4 was 2.11 ± 0.64,7.22 ± 1.13,11.16 ± 1.23 and 19.12 ± 3.16 (F =96.26,P ＜ 0.05),and the expression level of PDCD4 protein was 1.22 ± 0.08,2.09 ± 0.20,2.26 ± 0.1 1 and 2.37 ± 0.07 (F=75.37,P＜ 0.05),respectively,in HaCaT cells treated with metformin of 25,50,75 and 100 mmol/L.Similarly,no significant difference was observed between the control cells and those treated with 25 mmol/L metformin in the expression level of miR-21-5p mRNA,PDCD4 mRNA or protein,but decreased expression of miR-21-5p mRNA and increased expression of PDCD4 mRNA and protein were noted in cells treated with the other 3 concentrations of metformin compared with the control cells (all P＜ 0.05),and significant differences were also found in the expression levels of miR-21-5p mRNA as well as PDCD4 mRNA and protein among the 4 intervention groups (all P ＜ 0.05).Conclusion Metformin can markedly inhibit the proliferation of HaCaT cells in vitro,likely by downregulating miR-21-5p expression and upregulating PDCD4 expression.

0 05) Conclusion The COMT G1947→A gene polymorphism is not associated with the generation and the severity of PIH The mutation genotype does not increase the risk of PIH

Objective To screen for differentially expressed proteins in sera from patients with common types of psoriasis,and to identify plasma protein markers for psoriasis.Methods Serum samples were collected from 6 patients with progressive psoriasis vulgaris,5 patients with erythroderma psoriaticum,and 6 healthy human controls,and then pooled into 3 pools:psoriasis vulgaris pool,erythroderma psoriaticum pool and control pool.After removal of high-abundance albumin and IgG,the pooled samples were analyzed by two-dimensional electrophoresis (2-DE).An electrophoretic gel image analysis software was used to locate differentially expressed protein spots followed by peptide mass fingerprinting with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).The National Center for Biotechnology Information (NCBI) protein databases were then searched for the identification of differentially expressed proteins.Results All the three pooled serum samples were well seperated by 2-DE.As the gel image analysis software showed,there were 33 protein spots differentially expressed between the patients with psoriasis vulgaris and the healthy controls,17 between the patients with erythroderma psoriaticum and the healthy controls,and 26 between the patients with psoriasis vulgaris and those with erythroderma psoriaticum.Finally,14 proteins were identified as differentially expressed proteins.The patients with psoriasis vulgaris showed higher expression of complement component 3,interleukin-16,vitamin D-binding protein and α1-antitrypsin compared with the healthy controls; the patients with erythroderma psoriaticum showed increased expression of complement component 3,complement component H,α1-antitrypsin,hemopexin and haptoglobin,but decreased expression of serum amyloid protein compared with the healthy controls,as well as enhanced expression of α1-antitrypsin,complement component H,complement component 4 and haptoglobin compared with those with psoriasis vulgaris.Conclusion Differences exist in serum protein profiles between patients with psoriasis vulgaris and erythroderma psoriaticum,and healthy human controls.

Objective To investigate the association of eIF4E and MMP-9 gene polymorphisms with psoriasis vulgaris in Han population of Shandong province.Methods A population based case-control association study was carried out in 188 patients with psoriasis vulgaris and 280 healthy human controls of Han nationality from Shandong province.Taqman SNP genotyping assay was performed to assess three SNPs,including rs4810482 and rs3918254 in MMP-9 gene and rs11723037 in eIF4E gene.Pairwise linkage disequilibrium was evaluated by using Haploview 4.2 software,and the frequencies of alleles and genotypes were analyzed by using Plink 1.07 software.Results The frequency of rs4810482 T allele was significantly lower in patients with psoriasis vulgaris than in the normal human controls(OR =1.49,95％ CI:1.12-1.99,P ＜ 0.01),and the significant difference still remained under recessive and dominant model.Bioinformatic analysis revealed that the rs4810482 altered the binding site of transcription factor,while no association was observed between psoriasis and either of the other two SNPs.Conclusions The SNP rs4810482 located at the upstream regulatory region of MMP-9 gene is significantly associated with psoriasis,hence,MMP-9 gene may be a susceptibility gene for psoriasis in Han population of Shandong province.

Objective To discuss protein marks expressed differentially in placenta of Down's syndrome by means of proteomics. Methods We collected placenta of 18 patients(from March 2009 to December 2009 at Beijing Obstetrics and Gynecology Hospital), and divided them into two groups, one was 10 patients with fetal Down's syndrome, the other was normal pregnancies (normal chromosome) with other diseases. We separated proteins expressed in placentas of two groups by two-dimensional difference gel electrophoresis (2D-DIGE), and then analyzed the differential protein spots by software Decyder 6. 5, then,spots differentially expressed by 1.5 fold or more were analyzed by matrix assisted laser desorption ionizationtime of flight-mass spectrometry (MALDI-TOF-MS). In the end, the differential expressional levels of partially identified proteins were validated by western blot analysis. Results (1) Differential proteins of two groups protein spots of placentas separated by 2D-DIGE were analyzed by software Decyder 6. 5 (these colored lights scattered in the image were protein spots), a total of 56 spots out of 352 were differentially expressed (P<0. 05) in two groups. We analyzed 17 protein spots(12 protein spots were over-expressed and 5 protein spots were down-expressed) differentially expressed by 1.5 fold or more by MALDI-TOF-MS.(2) Protein matching after searching protein database, 17 protein spots turn out to be 10 proteins. Four kinds [superoxide dismutase 1 (SOD1), peroxiredoxin 6 (PRDX6), heat shock protein 27 (HSP27),endoplasmic reticulum protein 29 (ERP29)] of them were validated by western blot analysis, the group of fetal Down's syndrome were 0.74 ±0. 12,0.29 ±0. 10,0.53 ±0. 16,0.20 ±0. 09,the group of normal pregnancies were 0. 51 ±0. 08,0. 34 ± 0. 16,0. 18 ± 0. 07,0. 35 ± 0. 09, the results confirmed the observed changes in proteomics. Conclusions Compared with normal pregnancies, there were differential proteins expressed in placenta of Down's syndrome. This approach might provide new screening markers in use for prediction of Down's syndrome, however, further study should be done to make these 4 proteins (SOD1,HSP27, ERP29, PRDX6) be new screening markers.

Objective To investigate genes involved in the mechanisms underlying the progression of severe preeclampsia.Methods We conducted a muhiregional gene expression analysis using peripheral leucocytes from patients with preeclampsia and normal controls.Total RNA was extracted from peripheral blood of six severe preeclampsia and five normotensive pregnancies.We performed genome-wide expression profiling using Affymetrix HG_U133 plus 2.0 chips to screen out differentially expressed genes of 2 fold or more and q_value ＜ 5.4%.Using Gene Ontology we identified the function of differentially expressed genes after cluster analysis.Results Among the 47 000 genes that were screened in the microarray,140 genes were found to be differentially expressed between normal and preeclamptic pregnancies. Eighty six up-regulated candidate genes were mainly involved in cysteine metabolism urea cycle and metabolism of amiogroups,proteasome,TGF-beta signaling pathway, and the ratio of calponin2 (CNN2), matrix metallopeptidase 8 (MMP8),V-set and immunoglobulin domain containing 4 (VSIG4),proteasome 26S subunit ATPase 5 (PSMC5) was evidently increased in preeclampsia patients.Among 54 down-regulatedcandidates,natural killer cell mediated cytotoxicity,antigen processing and presentation,metabolism of xenobiotics by cytochrome P450 were the main pathways.KIR3DL2,AKR1C3,CHURC1 and SLC25A13 were obviously decreased in preeclampsia patients. Conclusions The gene expression of peripheral leucocytes in preeclampsia patients is significantly different from that of uncomplicated pregnancies.CNN2,MMP8,VSIG4,PSMC5,KIR3DL2,AKR1C3,CHURC1 and SLC25A13 may be involved in the molecular mechanisms underlying the progression of severe preeclampsia.

OBJECTIVETo investigate the immunological characteristics of human tonsil mesenchymal stem cells (TMSCs).

METHODSHuman tonsil tissues were obtained from the children patients with chronic tonsillitis. TMSCs were separated, cultured, and were detected the expression profiles of HLA-I, HLA-II, CD80, CD86 by flow cytometry. The measurement of immunogenicity, the effect on phytohemagglutinin (PHA) induced peripheral blood mononuclear cell (PBMCs) proliferation and mixed lymphocytes reaction (MLR) were performed to identify the immunological characteristics of TMSCs. The co-cultures of TMSCs + PBMCs + PHA and TMSCs + MLR were established, respectively, and the concentration of kynurenine, which is the metabolin of indoleamine 2, 3-dioxygenase, in the culture supernatant were examined. Then we added 1-methyl-L-tryptophan into the co-culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, and tested the proliferation of PBMCs. Each experiment was repeated three times, and there were six samples in each group. Statistical significance was assessed by analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant.

RESULTSTMSCs expressed HLA-I, were negative for HLA-II and co-stimulatory molecules CD80 and CD86. The stimulation index in the group of TMSCs + allogeneic PBMCs was 1.38 ± 0.26, whereas the stimulation index in the group of allogeneic PBMCs was 1.22 ± 0.28, and there was no significant difference between the two groups (P > 0.05), indicating that TMSCs could not initiate the proliferation of allogeneic PBMCs. The stimulation indexes in the group of TMSCs + allogeneic PBMCs + PHA were 1.49 ± 0.29 and 1.23 ± 0.22, respectively, whereas the stimulation index in the group of allogeneic PBMCs + PHA was 4.60 ± 0.81, and the difference between the two groups had a statistical significance (P < 0.05) suggesting that TMSCs could inhibit PHA-induced PBMCs proliferation. The stimulation indexes in the group of TMSCs + MLR were 1.29 ± 0.23 and 1.26 ± 0.27, respectively, however, the stimulation index in the group of MLR was 3.04 ± 0.66, and the difference between the two groups had a statistical significance (P < 0.05), demonstrating that TMSCs could suppress MLR-induced PBMCs proliferation. The levels of kynurenine were (26.0 ± 2.3) μmol/L and (23.5 ± 4.5) μmol/L in the culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, thus elevating significantly. After adding of 1-methyl-L-tryptophan, TMSCs-mediated-proliferation suppression of PBMCs restored to normal levels.

CONCLUSIONTMSCs possess low immunogenecity and immunosuppressive function, may be used in allogeneic transplantation.

Cell Proliferation ; Cells, Cultured ; Child ; Coculture Techniques ; Flow Cytometry ; Humans ; Immunosuppression ; Kynurenine ; analysis ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; Palatine Tonsil ; cytology ; Tryptophan ; administration & dosage ; analogs & derivatives

Objective To study the coexpression of protein folding modulators,including chaperones(DnaK,DnaJ,and GroEL-GroES) and disulfide isomerase Dsb molecules(DsbA and DsbC) on the bioactivity of eukaryotic expressed urokinase(UK).Methods The gene sequences of DnaK,DnaJ,GroEL-GroES,DsbA and DsbC were isolated from the chromosome of E.coli by PCR.The obtained fragments mentioned above were cloned into the plasmid pBAD-1 under the control of araB promoter and then the plasmids were transformed into E.coli BW25113 and HB101 together with a compatible vector pQE-UK.The amount of expressed UK was measured by Bradford method and its activity was analyzed by fibrin plate method for its fibrinolysis.Results The activity of expressed UK was increased by co-expressing with DnaK,GroEL-GroES,DsbA,and DsbC in BW25113.Among them,the highest activity was achieved by co-expressing with GroEL-GroES.Conclusion Co-expressing of chaperones or disulfide isomerase can improve the bioactivity of UK expressed in E.coli.

The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.